Antisense oligonucleotides (ASONs) are a promising new class of therapeutics designed to attenuate or correct the expression of discordantly expressed genes with high specificity and avidity. Efficiency of ASONs in cell culture is known for a long time. They have become nowadays an indispensable tool in biomedical research. Clinical proof of efficacy has been established with the approval of the first ASON therapeutic, fomivirsen sodium in 1998 (marketed as Vitravene™, Isis Pharmaceuticals).
At present, there are several clinical trials underway using antisense compounds directed at various targets playing mainly a role in cancer, viral diseases and inflammatory disorders either as single-agent or in combination with other related therapeutics. Yet, after more than three decades from the inception of antisense technology and in spite of numerous promising clinical trial reports, fomivirsen and mipomersen are the only antisense therapies on the market. Nevertheless, its route of administration, i.e. intravitreal injection, is indicative of its lack of appropriate pharmacokinetic properties. A major challenge in widespread use of ASONs is their yet inadequate uptake and biological stability for a sufficient length of time to achieve the desired effect. This has prompted the development of new generations of ASONs.
The first-generation modifications (first-generation ASONs) were primarily on the backbone of the ODN molecules, such as phosphorothioate oligodeoxynucleotides (PS-ODNs), methylphosphonates, and phosphoramidates. Like their native counterparts, PS-ODNs have a negatively charged backbone and are capable of supporting RNase H activity offering nuclease resistance sufficient for parenteral administration. At the same time, this simple chemical modification imparts favorable pharmacokinetic properties including improved tissue distribution, reduced urinary excretion, and prolonged residence time in tissues and cells. These intrinsic properties have made PS-ASONs the structure of choice as first-generation ASONs.
With respect to clinical application it can be stated that obviously the phosphorothioates “have made the race”. However, their widespread clinical application may be limited as a result of their rather poor pharmacodynamic and safety issues. Although concern about such issues has been raised since a long time, it was only recently confirmed that the thioate modification as a structural moiety is sequence-independently associated with an intrinsic apoptotic effect. Based on these results, the use of the phosphorothioates seems to be acceptable only in therapeutic applications where an apoptotic effect is desired or at least acceptable.
The second-generation ASONs represent oligonucleotides in which the structural modification is not limited to the backbone linkage but includes structural modifications of the nucleoside. They were designed to improve the efficacy of the ASONs, to increase the binding affinity, to enhance the nuclease resistance, to improve cellular absorption and to modulate the protein binding of oligonucleotides. The 2′-modification was evidently the winner within this generation. In 1991, the superior biological efficacy was demonstrated of 2′-O-methyl and 2′-O-ethyl oligoribonucleotides compared with the unmodified antisense RNA for studying snRNP-mediated pre-mRNA splicing and processing.
The degree of nuclease resistance conferred by a simple 2′-O-methyl substituent however does not completely suffice for the corresponding phosphodiester-based oligonucleotides to be useful for antisense application. The use of mixed backbone oligonucleotides which contain appropriately placed segments of phosphorothioate oligodeoxynucleotides and differently modified oligodeoxynucleotide or oligoribonucleotides in the intervening gap have shown improvements over PS-ODNs.
More recently, minimally-modified antisense ODNs have been introduced. These are phosphodiester ODNs protected by two to five PS residues at their 3′-end by two PS residues at their 5′-end against potential degradation by 5′-exonucleases, and by phosphorothioate linkages at internal pyrimidine nucleotides against degradation by endonucleases. Definitely, all these thioate-containing modifications have to be assessed in the light of the intrinsic thioate toxicity mentioned above.
The third-generation ASONs represent major structural changes and contain a variety of modifications within the ribose ring and/or the phosphate backbone. Such modifications were frequently carried out in the frame of basic scientific research to promote the understanding of the mechanism of base pairing.
Undoubtedly, the goal of most of these modifications has been to improve the bio-stability and cellular uptake, to optimize tissue and cell distribution for a particular molecular target as well as being less toxic.
At this stage it has to be mentioned that during the early phases of antisense research the mechanism of inhibition was not fully delineated. Several “effector” approaches have been proposed, e.g. ribozymes, intercalators, etc. In this respect, the finding that the enzyme RNase H is cleaving the sense strand efficiently has made most of the other approaches obsolete and has focused the attention on the RNase H compatibility of specific modifications. It is not surprising however that most of these third-generation modifications (such as PMOs and LNAs) render them RNase H-incompetent.
In summary, there is no doubt about the pharmacodynamic potential of the antisense principle. Nevertheless, it is evident that during all phases of research it was rather the emergence of novel approaches in nucleic acid therapy, like siRNA or miRNA that has been driving the progress, while the equally important aspects of poor bioavailability and weak pharmacokinetic parameters have not yet received the required attention.
To date, the implicit aim has been to make enzyme-resistant DNA analogues capable of forming most stable hybrids with complementary DNA or RNA. Many of such analogues are now at hand, now it is time to focus more on biological parameters such as cellular uptake, bioavailability, and general pharmacokinetics. Appropriate balance of these properties enables drug molecules to attain and maintain sufficient systemic and/or target concentrations to exert therapeutic effects through optimum absorption, distribution, metabolism, and excretion (ADME) processes. Due to the awareness that the rather high attrition rate of compounds, observed only in the development phase, is caused by unfavorable ADME and toxicity properties, more and more efforts are being put to the field of ADME.
Natural oligonucleotides have a phosphodiester backbone that is susceptible to degradation by abundant nucleases present in vivo. Hence, oligonucleotides must be chemically modified or protected by appropriate formulations in order to be used as therapeutic agents.
With regard to the ADME properties of various classes of the ASONs, it seems that these agents share a common dilemma, i.e. poor pharmacokinetic properties, particularly poor bioavailability and cellular uptake. One of the approaches implemented to improve the antisense activity of nucleic acids is the development of suitable delivery systems. In cell culture studies, uptake-enhancing agents such as cationic lipids or polymers are routine tools to transfect the cells. However, in vivo co-administration of oligonucleotides with these agents has proved problematic owing to their unfavorable pharmacokinetics and considerable toxicity.
Another promising approach pursued to improve antisense activity relies on performing minor structural changes designed to alter multiple properties of the oligonucleotide, for instance, improving nuclease resistance, hybridization and cellular uptake of the oligonucleotide at the same time. Hence, a variety of chemical modifications have been designed including alterations in the backbone chemistry, modifications on the 2′-position of the ribofuranose ring, altered ring structure, conjugation with other molecules or oligomers, nucleobase modifications and others.
Rajur, S B et al. (Rajur, S B et al. Bioconjugate chemistry, 1997, 8(6), pp 935-940) describes a conjugate of an antisense oligonucleotide having a thiol linker to asiaglycoprotein via a disulfide bond.
In Lee, S H et al. (Lee, S H et al. Macromolecular Bioscience, 2010, 11(3), pp 410-418), a conjugate of a siRNA having a thiol linker to polyethylene glycol via a disulfide bond is described.
US 2010/190691 A1 describes a conjugate of siRNA having a thiol linker to cell penetrating peptides via a disulfide bond.
In U.S. Pat. No. 6,153,737 A a conjugate of a phosphorothioate oligonucleotide having a thiol linker at a 2′ position to cholesterol via a disulfide bond is described.
Shengxi, J et al. (Shengxi, J et al. The Journal of organic chemistry, 2005, 70(11), pp 4284-4299) discloses nucleoside phosphoramidites for solid-phase synthesis with protected amine or thiol functional group. The nucleoside phosphoramidites either include each of the four common RNA nucleotides (U, C, A, and G) with a 2′-(2-aminethoxy)-2′-deoxy substitution or include each of the four common RNA nucleotides (U, C, A, and G) with an 2′-(2-mercaptoethoxy)-2′-deoxy substitution (i.e., a tethered 2′-thiol). Goodwin, J T et al. (Goodwin, J T et al. Journal of the American Chemical Society, 1996, 118(22), pp 5207-5215) discloses cross linked RNA, wherein the cross-linked RNA is achieved by two 2′-O-alkylthiol modified cytosine residues.
Gundlach, C W et al. (Gundlach, C W et al. Tetrahedron Letters, 1997, 38(23), pp 4039-4042) describes guanosine analogs exhibiting an alkylthiol substituent at the 2′-hydroxyl of the ribose.